The lonq term objective of the proposed research is to understand the critical early events in the initiation and regulation of blood platelet function in hemostasis. Evidence is increasing that platelets need to be activated in seconds or fractions of a second to prevent excessive bleeding from rapidly-flowing blood. Biochemical changes, such as an increase in cellular calcium, phospholipid hydrolysis, or phosphorylation of 1 specific proteins, have been detected within 0.5 sec of platelet activation by ADP or thrombin. However, important questions exist as to the regulation and mechanisms leading to platelet "shape change", aggregation and secretion. Perhaps the most significant platelet activator is ADP, and how it initiates siqnal transduction is not well understood. The sources of necessary intracellular calcium are not clear, or whether phosphatidylinositol hydrolysis is necessarily linked to the early ( 2 sec) major rise in cytoplasmic Ca++. Potential changes in intracellular location of some important enzymes such as protein kinase C, are not known during the first 5 sec after activation, or the functional consequences of such chanqes. Platelet function will be analyzed by a new quenched-flow approach which enables precise control of reaction times down to about 0.1 sec after initiation. Aims are to test the hypothesis that turnover of phosphatidylinositol is requried for the rapid ( 2 sec) increase in intracellular calcium caused by ADP, in contrast to thrombin. Special interest will be directed to potential ADP-induced lipid hydrolysis and corresponding formation of the cyclic or non-cyclic inositol phosphates. The importance of extracellular Na+ and cyclic GMP in the regulation of rapid calcium fluxes will be assessed. An important member of a new group of four phosphoproteins (185,140 104 and 77 kDa) will be studied, since their kinetics and very early phosphorylation (0.3 sec) are quite distinct from the well-defined 20 and 47 kDa proteins. We will test by immuno-blottinq procedures the hypothesis that the 77 kDa protein is protein kinase or possibly caldesmon, ADP will serve as major agonist, but thrombin will also be used as a classic stimulator of phosphatidylinositol turnover, Lastly, using a new sprayfreezing method coupled to immuno-electromicroscopy, we propose to study the intra- platelet localization-and functional consequences of protein kinase C during the first few seconds after platelet activation.